Cell adhesion molecules (abbreviated CAMs) are neuronal cell surface glycoproteins which help to mediate the cohesive interactions between developing or regenerating neurities. It is believed that proper adhesion of neuronal cells to each other and to their surrounding extracellular matrix is essential for maintaining and/or promoting growth of the neuronal cells.
A number of cell adhesion molecules have been isolated and identified, including neural cell adhesion molecule (N-CAM), nerve growth factor-inducible large external glycoprotein (NILE), neuron-glial CAM (Ng-CAM) and closely related proteins L1 (L1 antigen). These integral membrane glycoproteins, or molecules closely related thereto, have been described by Applicants and others in the nervous system of several species.
Representative examples of such identification and description include L1cam in mouse, (Rathjen, F. G., Schachner, M., Immunocytological And Biochemical Characterization Of A New Neuronal Cell Surface Component (L1 Antigen) Which Is Involved In Cell Adhesion. EMBO J. 3:1-10 (1984)); NILE in rat, (McGuire, J. C., Greene, L. A., Furano, A. V., Nerve Growth Factor Stimulates Incorporation Of Fucose Or Glucosamine Into An External Glycoprotein In Cultures Rat PC12 Pheochromocytoma Cells. Cell 15:357-365 (1978)); Ng-CAM/8D9/G4 in chick, (Grumet, M., Edelman, G. M., Neuron-Glia Cell Adhesion Molecule Interacts with Neurons and Astroglia via Different Binding Mechanisms. J. Cell Biol. 106:487-503 (1988); and, Lemmon, V., McLoon, S., The Appearance Of An L1-Like Molecule In The Chick Primary Visual Pathway, J. Neurosci. 6:2987-2994, (1986)); and Neuroglian in Drosophila (Bieber, A. J., Snow, P. M., Hortsch, M., Patel, N. H., Jacobs, J. R., Traquina, Z. R., Schilling, J., Goodman, C. S., Drosophila Neuroglian: A Member Of The Immunoglobulin Superfamily With Extensive Homology To The Vertebrate Neural Adhesion Molecule L1. Cell 59:447-460 (1989)).
These molecules share similar biochemical properties, immunological crossreactivity, localization predominantly on axons of projection neurons, homology in nucleotide sequence as well as functional similarity.
The L1 cell adhesion molecule, which was first isolated and characterized in mouse (i.e. L1cam) is a membrane-spanning glycoprotein that has sequence similarity with both fibronectin and the immunoglobulin superfamily. Specifically, L1 cell adhesion molecules possessing six extracellular Ig-like domains, three to five fibronectin (Fn) type III-like repeats, a transmembrane segment, and a small cytoplasmic region. The membrane-spanning region links the extensive extracellular domain to the substantial cytoplasmic domain.
Several lines of evidence suggest that the L1 cell adhesion molecule plays an important role in neuronal growth and fasciculation. First, it is expressed by subpopulations of neurons in the central nervous system and on nerons and Schwann cells in the peripheral nervous system. This expression is early on in developing (Martini, R., Schachner, M., Immunoelectron Microscopic Localization Of Neural Cell Adhesion Molecules (L1, N-CAM, MAG) And Their Shared Carbohydrate Epitope And Myelin Basic Protein (MBP) In Developing Sciatic Nerve. J. Cell Biol. 103:2439-2448 (1986)) and regenerating axons (Daniloff, J. K., Chuong, C.-M., Levi, G., Edelman, G. M., Differential Distribution Of Cell Adhesion Molecules During Histogenesis Of The Chick Nervous System. J. Neurosci. 6:739-758 (1986); and, Martini, R., Schachner, M., Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, MAG) and their shared carbohydrate epitope and myelin basic protein (MBP) in developing sciatic nerve. J. Cell Biol. 103:2439-2448 (1986)).
Secondly, since antibodies to L1 disrupt fascicle formation in vitro (Stallcup, W. B., Beasley, L., Involvement Of The Nerve Growth Factor-Inducible Large External Glycoprotein (NILE) In Neurite Fasciculation In Primary Cultures Of Rat Brain. Proc. Natl. Acad. Sci. USA 82:1276-1280 (1985)) and in vivo (Landmesser, L., Dahm, L., Schultz, K., Rutishauser, U., Distinct Roles For Adhesion Molecules During Innervation Of Embryonic Chick Muscle. Dev. Biol. 130:645-670 (1988)), L1 participates in axonal fasciculate formation.
Finally, it has been shown by Applicants and others that purified L1 is a potent substrate for neurite growth and development (Lagenaur, C., Lemmon, V., A L1-Like Molecule, The 8D9 Antigen, Is A Potent Substrate For Neurite Extension. Proc. Natl. Acad. Sci. USA 84:7753-7757 (1987)).
Moreover, a number of observations are consistent with the existence of a L1 human homologue. Human tumors, especially neuroblastoma, demonstrate immunoreactivity to L1 antibodies (Mujoo, K., Spiro, R. C., Reisfeld, R. A., Characterization Of A Unique Glycoprotein Antigen Expressed On The Surface Of Human Neuroblastoma Cells. J Biol. Chem. 261:10299-10305 (1986); and, Figarella-Branger, D. F., Durbec, P. L., Rougon, G. N., Differential Spectrum Of Expression Of Neural Cell Adhesion Molecule Isoforms And L1 Adhesion Molecules On Human Neuroectodermal Tumors. Cancer Research 50:6364-6370 (1990)). In addition, Biochemical analysis of a glycoprotein isolated from human brain using an anti-neuroblastoma monoclonal antibody revealed that the antigen was very similar to mouse L1cam, Wolff, J. M., Frank, R., Mujoo, K., Spiro, R. C., Reisfeld, R. A., Rathjen, F. G., A human Brain Glycoprotein Related To The Mouse Cell Adhesion Molecule L1. J. Biol Chem. 263:11943-11947 (1988)). To conform with the HGMW approved nomenclative, (Human Gene Mapping Workshop, published 1987 in Cytogenet. Cell Genet., Vol. 46, pages 11-28) mouse L1 cell adhesion molecule is referred hereinafter as L1cam and human L1 cell adhesion molecule is referred to as L1CAM.
Recently, partial sequences obtained for a human genomic clone, Djabali, M., Mattei, M. G., Nguyen, C., Roux, D., Demengeot, J., Denizot, F., Moos, M., Schachner, M., Goridis, C., Jordan, B. R., The Gene Encoding L1, A Neural Adhesion Molecule Of The Immunoglobulin Family, Is Located On The X-Chromosome In Mouse And Man, Genomics 7:587-593 (1990) and a human melanoma cDNA clone (Harper, J. R., Prince, J. T., Healy, P. A., Stuart, J. K., Nauman, S. J., Stallcup, W. B., Isolation And Sequence Of Partial cDNA Clones of Human-L1--Homology Of Human And Rodent-L1 In The Cytoplasmic Region, J. Neurochem. 56:797-804 (1991) confirmed that a human L1-like molecule exists.
Further studies localized the gene for human L1 to the q28 band on the X chromosome (Djabali, M., Mattei, M. G., Nguyen, C., Roux, D., Demengeot, J., Denizot, F., Moos, M., Schachner, M., Goridis, C., Jordan, B. R., The Gene Encoding L1, A Neural Adhesion Molecule Of The Immunoglobulin Family, Is Located on the X-Chromosome in Mouse and Man, Genomics 7:587-593 (1990), the homologous region to the A6-B region of the mouse X chromosome where the L1cam gene is located.
The knowledge that a L1-like molecule exists in humans leads to the conclusion that L1CAM may be important in promoting axon regeneration in trauma or disease states of the human nervous system. Therefore, the Applicants have isolated and purified L1 from human brain and conducted in vitro experiments on the natural substance that demonstrate that human L1CAM, like chick and mouse L1cam, can support neuron attachment and neurite growth.
In addition, Applicants have cloned and sequenced cDNAs encompassing the entire coding region of L1CAM. This information will allow future studies on the structure and function of L1CAM and permit the construction of cell lines expressing L1CAM for in vitro and in vivo experiments on nerve growth and regeneration.
These and other objects and features of the invention will be apparent from the following description and from the claims.